The major goal of this application is to evaluate the phenomenon of sperm DNA degradation observed as the result of various sperm treatments. The hypothesis is that there exists a mechanism in mammalian fertilization that prevents the transmission of potentially damaged DNA to the embryo by disruption of the paternal chromosomes that this mechanism is based on the activity of endogenous nucleases, and that it can be active within sperm cells. In our preliminary studies we have shown that when spermatozoa are treated with various chemicals (detergents, DTT, exogenous DNA) or are subjected to mechanical insults (freeze drying and freezing without cryoprotection), and then injected into the oocytes, the paternal chromosomes in the zygotes are broken. We have also shown that this DNA degradation could be partially or completely prevented when ion chelators, EGTA or/and EDTA, were present in the sperm handling medium, which suggests the involvement of endogenous nuclease/s in this process. The idea that spermatozoa may have nuclease-dependent mechanism for DNA degradation differs from current thinking on the function of mammalian male gametes. In this application we will initiate the exploration on the origin and function of sperm DNA damage by expanding our preliminary data and answering the first major questions that this data brought. In Specific Aim 1, we will determine the causes of sperm DNA damage, and whether they are inherent to the cell or, are the result of experimental manipulation. We will explore what are the conditions under which the paternal chromosome breaks, examine early post-fertilization events after ICSI with treated spermatozoa, and test if paternal chromosome breakage can be observed in the absence of ICSI, to exclude the possibility that observed chromosome breakage is an artifact of ICSI combined with manipulation. Specific Aim 2 will answer the most crucial question of this application: Does DNA degradation occur in the spermatozoa or in the oocyte? We will test whether paternal DNA breakage can be induced in the oocytes after fertilization. We will analyze sperm DNA integrity before and after DNA synthesis. We will also evaluate if maternal (oocyte) DNA is degraded in the result of insults to which sperm, oocyte, or both are exposed. Finally, we will establish whether spermatozoa are able to induce degradation of DNA other than their own, a direct test to confirm the possibility that DNA degradation mechanism exist within sperm heads. In Specific Aim 3, we will test if paternal DNA degradation depends on the activity of endogenous Ca2+ and Mg2+dependent nucleases or topoisomerase II. We will establish if stresses to which spermatozoa are exposed can be neutralized by the presence of on chelators and other nuclease inhibitors, what is the range of nuclease activity, and what are the conditions under which the maximum protection is provided. We will also establish if endogenous Ca2+ and Mg2+dependent nucleases are present in the oocytes, if they can be inhibited there, and what will be the effects of this inhibition on paternal DNA integrity. The significance of this proposal is that it will test a novel idea that spermatozoa are active cells able to respond to their environment. This response is suspected to be a part of the mechanism that allows preventing the transmission of potentially damaged DNA to the embryo during fertilization. The study will have impact on basic science by advancing our understanding of biological system of mammalian reproduction. It will also have significance for clinical research and health and welfare of infertile couples providing clues for human assisted reproduction clinics as to what treatments evoke DNA-degrading mechanisms in spermatozoa and therefore should be avoided.